MERS-CoV vaccine with trimeric S1-CD40L fusion protein

ABSTRACT

An immunogenic CD40-targeted trimeric MERS-CoV S1 fusion polypeptide as well as a corresponding polynucleotide encoding it and its use for safely inducing immune responses directed against MERS-CoV without inducing vaccine associated respiratory pathologies associated with non-targeted vaccines.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.17/101,572, pending, having a filing date Nov. 23, 2020 which is adivisional of U.S. application Ser. No. 16/201,409, filed Nov. 27, 2018(now U.S. Pat. No. 10,849,972) which is incorporated by reference.

BACKGROUND Field of the Invention

The invention falls within the fields of molecular virology, immunologyand medicine.

Description of the Related Art

The “background” description provided herein is for the purpose ofgenerally providing a context of the disclosure. Work of the presentlynamed inventor, to the extent it is described in this background sectionas well as aspects of the description which may not otherwise qualify asprior art at the time of filing are neither expressly or impliedlyadmitted as prior art against the present invention.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is arecently emergent zoonotic virus that was first isolated from a fatalhuman infection in Saudi Arabia in 2012; Zaki, A. M. et al. Isolation ofa novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl.J. Med. 367, 1814-1820 (2012).

This zoonotic virus causes a variety of clinical manifestations inhumans ranging from asymptomatic or mild infections to severe acute andfatal respiratory symptoms associated with fever, cough, acutepneumonia, shortness of breath, systemic infection and occasionalmulti-organ failure; Zaki et al., id. (2012).

As of January 2018, MERS-CoV has caused more than 2,143laboratory-confirmed infections in 27 countries with a 30-40% mortalityrate; Saudi Arabia is the most affected country; World HealthOrganization. Middle East respiratory syndrome coronavirus(MERS-CoV)—Saudi Arabia. Disease outbreak news 26 Jan. 2018 (hypertexttransferprotocol://worldwideweb.who.int/csr/don/26-january-2018-mers-saudi-arabia/en/).

Most MERS-CoV cases have been linked to local infections ortransmissions that occur in countries within the Arabian Peninsula whereMERS-CoV has been endemic for more than six years. However, MERS-CoV hasthe potential to spread globally as apparent from a 2015 outbreak inSouth Korea; Park, S. H. et al. Outbreaks of Middle East RespiratorySyndrome in Two Hospitals Initiated by a Single Patient in Daejeon,South Korea. Infect. Chemother. 48, 99-107 (2016).

Countries in or near the Arabian Peninsula with laboratory-confirmedMERS cases include Bahrain, Iran, Jordan, Kuwait, Lebanon, Oman, Qatar,Saudi Arabia, United Arab Emirates (UAE), and Yemen. Countries outsidethe Arabian peninsula with travel-related MERS-CoV infections includeAlgeria, Austria, China, Egypt, France, Germany, Greece, Italy,Malaysia, Netherlands, Philippines, Republic of Korea, Thailand,Tunisia, Turkey, United Kingdom (UK), and the United States of America(USA) hypertext transfer protocolsecure://worldwideweb.cdc.gov/coronavirus/mers/index.html, last accessedAug. 17, 2018, incorporated by reference).

According to the World Health Organization (WHO) people at increasedrisk of MERS-CoV infection include those who live in or travel to theArabian peninsula, those in close contact with a person exposed to orinfected with MERs-CoV, and those in close contact with cameloids orcameloid products such as camel meat or milk. People at risk for severeMERS-CoV infection include those with diabetes, kidney failure, orchronic lung disease, and people who have weakened immune systems(hypertext transfer protocolsecure://worldwideweb.cdc.gov/coronavirus/mers/risk.htm (last accessed,Aug. 17, 2018; incorporated by reference).

MERS-CoV is a lineage C betacoronavirus and is closely related to batCoVs (HKU4 and HKU5); Zaki et al.; and Memish, Z. A. et al. Middle Eastrespiratory syndrome coronavirus in bats, Saudi Arabia. Emerg. Infect.Dis. 19, 1819-1823 (2013). Several lines of evidence suggest thatinsectivorous bats are the original source of MERS-CoV and that MERS-CoVmight have emerged via genetic recombination events; Memish et al.;Cotten, M. et al. Full-genome deep sequencing and phylogenetic analysisof novel human betacoronavirus. Emerg. Infect. Dis. 19, 736 (2013);Corman, V. M. Rooting the phylogenetic tree of MERS-Coronavirus bycharacterization of a conspecific virus from an African Bat. J. Virol.88, 11297-11303 (2014); and Anthony, S. J. et al. Further Evidence forBats as the Evolutionary Source of Middle East Respiratory SyndromeCoronavirus. MBio. 8, e00373-17 (2017). However, due to the limiteddirect contact between humans and bats, it is unlikely that bats are asource of primary human infection.

Molecular and serological epidemiological studies have shown awidespread detection of MERS-CoV in dromedary camels in the ArabianPeninsula and Africa suggesting that dromedaries are natural reservoirsof MERS-CoV or at least an important vector in its transmission tohumans; Perera, R. A. et al. Seroepidemiology for MERS coronavirus usingmicroneutralisation and pseudoparticle virus neutralisation assaysreveal a high prevalence of antibody in dromedary camels in Egypt, June2013. Euro. Surveill. 18, pii=20574 (2013); Reusken, C. B. et al. MiddleEast respiratory syndrome coronavirus neutralising serum antibodies indromedary camels: a comparative serological study. Lancet Infect Dis.13, 859-866 (2013); Meyer, B. et al. Antibodies against MERS coronavirusin dromedary camels, United Arab Emirates, 2003 and 2013. Emerg. Infect.Dis. 20, 552-559 (2014); Alagaili, A. N. et al. Middle East respiratorysyndrome coronavirus infection in dromedary camels in Saudi Arabia.MBio. 5, pii:e00884-14 (2014); and Azhar, E. I. et al. Evidence forcamel-to-human transmission of MERS coronavirus. N. Engl. J. Med. 370,2499-2505 (2014).

The Middle East respiratory syndrome-related coronavirus (MERS-CoV), isa novel positive-sense, single-stranded RNA virus of the genusBetacoronavirus. HCoV-EMC/2012 or Human Coronavirus Erasmus MedicalCenter/2012 (Accession/version: JX869059.2) is the name of a novelstrain of coronavirus first isolated from the sputum of an infectedperson. It was later named Middle East respiratory syndrome coronavirusor MERS-CoV. The polynucleotide sequences, locations of genes, codingsequences, and ORFS, and corresponding polypeptide sequences of MERS-CoVare incorporated by reference to the accession/version number above.

Unlike SARS-CoV, another coronavirus pathogen which uses humanangiotensin-converting enzyme 2 (ACE2) as its receptor for binding toACE2-expressing cells, MERS-CoV utilizes a different receptor,dipeptidyl peptidase 4 (DPP4) for binding to DPP4-expressing cells viaits Spike protein.

Spike protein is a type I membrane protein that is expressed as a trimeron the virus' surface. It typically has 1353 amino acid residues and ismade up of S1 and S2 subunits. The globular S1 subunit (residues 1-751)mediates virus binding to the DPP4 host cell receptor via its receptorbinding domain (RBD). The RBD is typically designated as amino acidresidues 367-606. The S2 subunit (residues 752-1353) contains anexternal domain, a transmembrane domain and a cytoplasmic domain, andmediates virus-cell membrane fusion as shown by FIG. 1; Lu, G. et al.Molecular basis of binding between novel human coronavirus MERS-CoV andits receptor CD26. Nature. 500, 227-231 (2013); and Raj, V. S. et al.Dipeptidyl peptidase 4 is a functional receptor for the emerging humancoronavirus-EMC. Nature. 495, 251-254 (2013).

MERS-CoV RBD consists of a core subdomain and a receptor-binding motif(RBM) (receptor binding subdomain) (residues 484 to 567) which containsthe motif and key residues to bind DPP4 receptor on the host cellmembrane.

The core subdomain is composed of five-stranded antiparallel β sheets(β1, β2, β3, β4 and β9) with two short a helices. The receptor-bindingsubdomain is a four-stranded antiparallel β sheet (ρ5, β6, β7 and β8),located between strands β4 and β9 of the core subdomain. A total of 14residues of MERS-CoV contact with 15 residues of the DPP4; Wang N, ShiX, Jiang L, et al. Structure of MERS-CoV spike receptor-binding domaincomplexed with human receptor DPP4. Cell Res. 2013 August; 23(8):986-93.doi: 10.1038/cr.2013.92; Chen Y, Rajashankar K R, Yang Y, et al. Crystalstructure of the receptor-binding domain from newly emerged Middle Eastrespiratory syndrome coronavirus. J Virol 2013; 10777-83.27.

In other coronaviruses, such as SARS-CoV, the S protein has been shownto be the main target for host immune responses and a target for SARSvaccines that led to robust induction of neutralizing antibodies(nAbs)-mediated protection in immunized animals; He, Y. et al.Receptor-binding domain of SARS-CoV spike protein induces highly potentneutralizing antibodies: implication for developing subunit vaccine.Biochem. Biophys. Res. Commun. 324, 773-781 (2004); He, Y. et al.Identification and characterization of novel neutralizing epitopes inthe receptor binding domain of SARS-CoV spike protein: revealing thecritical antigenic determinants in inactivated SARS-CoV vaccine.Vaccine. 24, 5498-5508 (2006); and Du, L. et al. The spike protein ofSARS-CoV—a target for vaccine and therapeutic development. Nat RevMicrobiol. 7, 226-236 (2009).

Likewise, the MERS-CoV S protein has been a focus for many MERS-CoVvaccine candidates. Several vaccine candidates based on full-length ortruncated S protein including vectored vaccines, DNA vaccines,nanoparticle vaccines, S or RBD protein-based subunit vaccines and wholeinactivated vaccines (WIV) have been developed and investigated inanimal models; Wang, L. et al. Evaluation of candidate vaccineapproaches for MERS-CoV. Nat. Commun. 6, 7712 (2015); Muthumani, K. etal. A synthetic consensus anti-spike protein DNA vaccine inducesprotective immunity against Middle East respiratory syndrome coronavirusin nonhuman primates. Sci. Transl. Med. 7, 301ra132 (2015); Coleman, C.M. et al. Purified coronavirus spike protein nanoparticles inducecoronavirus neutralizing antibodies in mice. Vaccine. 32, 3169-3174(2014); Du, L. et al. A truncated receptor-binding domain of MERS-CoVspike protein potently inhibits MERS-CoV infection and induces strongneutralizing antibody responses: implication for developing therapeuticsand vaccines. PLoS. One. 8, e81587 (2013); Al-Amri, S. S. et al.Immunogenicity of Candidate MERS-CoV DNA Vaccines Based on the SpikeProtein. Sci Rep. 7, 44875 (2017); Ma, C. et al. Searching for an idealvaccine candidate among different MERS coronavirus receptor-bindingfragments—the importance of immunofocusing in subunit vaccine design.Vaccine. 32, 6170-6176 (2014); Song, F. et al. Middle East respiratorysyndrome coronavirus spike protein delivered by modified vaccinia virusAnkara efficiently induces virus-neutralizing antibodies. J. Virol. 87,11950-11954 (2013); Haagmans, B. L. et al. An orthopoxvirus-basedvaccine reduces virus excretion after MERS-CoV infection in dromedarycamels. Science. 351, 77-81 (2016); Guo, X. et al. Systemic and mucosalimmunity in mice elicited by a single immunization with human adenovirustype 5 or 41 vector-based vaccines carrying the spike protein of MiddleEast respiratory syndrome coronavirus. Immunology. 145, 476-484 (2015);Kim, E. et al. Immunogenicity of an adenoviral-based Middle EastRespiratory Syndrome coronavirus vaccine in BALB/c mice. Vaccine. 32,5975-5982 (2014); Alharbi, N. K. et al. ChAdOx1 and MVA based vaccinecandidates against MERS-CoV elicit neutralising antibodies and cellularimmune responses in mice. Vaccine. 35, 3780-3788 (2017); and Agrawal, A.S. et al. Immunization with inactivated Middle East Respiratory Syndromecoronavirus vaccine leads to lung immunopathology on challenge with livevirus. Hum. Vaccin. Immunother. 12, 2351-2316 (2016).

Various MERS-CoV vaccine platforms to combat MERS-CoV have beeninvestigated. Most of these experimental vaccines were based on MERS-CoVfull-length or truncated versions of the spike protein; these prototypevaccines induced high levels of nAb (neutralizing antibody) andsometimes conferred complete protection against MERS-CoV challenge inseveral animal models. However, serious safety concerns are associatedwith vaccines for several CoVs including SARS-CoV and MERS-CoV includinginflammatory and immunopathological effects such as eosinophilicinfiltration of the lungs as well as Ab-mediated disease enhancement(ADE) in immunized animals upon viral challenge. Some of theseside-effects are shared with vaccines to other coronaviruses; Agrawal,et al., id.; Weingartl, H. et al. Immunization with MVA-basedrecombinant vaccine against SARS is associated with enhanced hepatitisin ferrets. J. Virol. 78, 12672-12676 (2004); Tseng, C. T. et al.Immunization with SARS coronavirus vaccines leads to pulmonaryimmunopathology on challenge with the SARS virus. PloS One. 7, e35421(2012); Yang, Z. Y. et al. Evasion of antibody neutralization inemerging severe acute respiratory syndrome coronaviruses. Proc. Natl.Acad. Sci. USA. 102, 797-801 (2005); Czub, M., Weingartl, H., Czub, S.,He, R. & Cao, J. Evaluation of modified vaccinia virus ankara basedrecombinant SARS vaccine in ferrets. Vaccine. 23, 2273-2279 (2005);Deming, D. et al. Vaccine efficacy in senescent mice challenged withrecombinant SARS-CoV bearing epidemic and zoonotic spike variants. PLoSMed. 3, 2359-75 (2006); Olsen, C. W., Corapi, W. V., Ngichabe, C. K.,Baines, J. D. & Scott, F. W. Monoclonal antibodies to the spike proteinof feline infectious peritonitis virus mediate antibody-dependentenhancement of infection of feline macrophages. J. Virol. 66, 956-965(1992); Jaume, M. et al. SARS CoV subunit vaccine: Antibody-mediatedneutralisation and enhancement. Hong Kong Med. J. 18 Suppl 2, 31-36(2012); Weiss, R. C. & Scott, F. W. Antibody-mediated enhancement ofdisease in feline infectious peritonitis: Comparisons with denguehemorrhagic fever. Comp. Immunol. Microbiol. Infect Dis. 4, 175-189(1981), each incorporated herein by reference in their entirety.

The inventor considered that induction of antibodies that do notneutralize MERS-CoV could increase the risk of undesired inflammatory orimmunological side-effects while having little or no effect on virusattachment. This is because epitopes outside of the S1 RBD of MERS-CoVcould induce anti-virus immune responses that do not block attachmentand entry of the virus into a host cell but which contribute toundesired inflammatory phenomena. Thus, use of a neutralizingepitope-rich S1 region was considered an attractive target for effectiveand safe MERS vaccine that blocks virus entry into host cells; Olsen etal., id.; Jaume et al., id.; and Weiss et al., id. However, even immuneresponses to S1 antigen may induce undesired side-effects.

Consequently, the inventor investigated whether targeting S1 antigen toa subset of antigen presenting cells could reduce undesirable immuneresponses to S1 and other Mers-CoV antigens while inducing a robustresponse against key portions of MERS-CoV involved in the host-parasiteinteraction.

CD40 is a receptor constitutively expressed on many antigen presentingcells. A ligand that binds to CD40 is CD40 Ligand or CD40L. CD40L is acostimulatory molecule that is an essential regulator of the immunesystem. Structurally CD40L is a type II membrane that is expressed onactivated CD4′ T cells and some other antigen presenting cells typicallyin a transient fashion; van Kooten, C. & Banchereau, J. CD40-CD40ligand. J. Leukoc. Biol. 67, 2-17 (2000); and Ma, D. Y. & Clark, E. A.The role of CD40 and CD154/CD40L in dendritic cells. Sem. Immunol. 21,265-272 (2009). Other ligands for CD40 that may be employed along withor instead of CD40L include antibodies or antibody fragments thatactivate or agonize CD40 including monoclonal antibodies and theirfragments or chimeric or fully human antibodies to CD40 and theirfragments, as well as other CD40 agonists or other molecules thatcrosslink CD40, including biosimilars of the aforementioned agents.

Based on the study of mutations in CD40 or CD40L genes in both animalsand humans, CD40L and its receptor CD40 together form a crucial linkbetween innate and adaptive immunity; van Kooten et al; Ma et al.;Bishop, G. A. & Hostager, B. S. The CD40-CD154 interaction in B cell Tcell liaisons. Cytokine Growth Factor Rev. 14, 297-309 (2003); Fujii, S.I., Liu, K Smith, C., Bontio, A. J. & Steinman, R. M. The linkage ofinnate to adaptive immunity via maturing dendritic cells in vivorequires CD40 ligation in addition to antigen presentation and CD80/86costimulation. J. Exp. Med. 199, 1607-1618 (2004); Allen, R. C. et al.CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome.Science. 259, 990-993 (1993); Kawabe, T. et al. The immune responses inCD40-deficient mice: impaired immunoglobulin class switching andgerminal center formation. Immunity. 1, 167-178 (1994); and Renshaw, B.R. et al. Humoral immune responses in CD40 ligand-deficient mice. J.Exp. Med. 180, 1889-1900 (1994).

Use of CD40L as a molecular adjuvant has been investigated by severalgroups using multiple strategies such as co-delivery of CD40L along withan antigen or retargeting the delivery vectors to CD40 on APCs; Cao, J.et al. CD40 ligand expressed in adenovirus can improve theimmunogenicity of the GP3 and GP5 of porcine reproductive andrespiratory syndrome virus in swine. Vaccine. 28, 7514-7522 (2010);Gómez, C. E., Nájera, J. L., Sánchez, R., Jiménez, V., & Esteban, M.Multimeric soluble CD40 ligand (sCD40L) efficiently enhances HIVspecific cellular immune responses during DNA prime and boost withattenuated poxvirus vectors MVA and NYVAC expressing HIV antigens.Vaccine. 27, 3165-3174 (2009); Huang, D. et al. Significant alterationsof biodistribution and immune responses in Balb/c mice administered withadenovirus targeted to CD40(+) cells. Gene Ther. 15, 298-308 (2007);Lin, F. C., Peng, Y., Jones, L. A., Verardi, P. H. & Yilma, T. D.Incorporation of CD40 ligand into the envelope of pseudotypedsingle-cycle simian immunodeficiency viruses enhances immunogenicity. J.Virol. 83, 1216-1227 (2009); Yao, Q. et al. Immunogenicity andprotective efficacy of a DNA vaccine encoding a chimeric protein ofavian influenza hemagglutinin subtype H5 fused to CD154 (CD40L) in Pekinducks. Vaccine. 28, 8147-8156 (2010); Hashem, A. M. et al. CD40 ligandpreferentially modulates immune response and enhances protection againstinfluenza virus. J. Immunol. 193, 722-734 (2014); and Fan, X. et al.Targeting the HA2 subunit of influenza A virus hemagglutinin via CD40Lprovides universal protection against diverse subtypes. Mucosal Immunol.8, 211-220 (2015), each incorporated herein by reference in theirentirety.

The inventor previously developed a non-replicating recombinantadenovirus-5 (rAd5) vectored prototype vaccine in which secretedinfluenza viral proteins are targeted to CD40-expressing APCs usingCD40L; Hashem et al., id.; and Fan et al., id. While viral vectors suchas rAd are effective and immunogenic, they are often associated withsafety problems. At least three groups have developed and examineddifferent rAd vectored MERS-CoV vaccines; Guo et al; Kim et al; Alharbi,N. K. et al. ChAdOx1 and MVA based vaccine candidates against MERS-CoVelicit neutralising antibodies and cellular immune responses in mice.Vaccine. 35, 3780-3788 (2017); and Munster, V. J. et al. Protectiveefficacy of a novel simian adenovirus vaccine against lethal MERS-CoVchallenge in a transgenic human DPP4 mouse model. NPJ Vaccines. 2, 28(2017). However, these previous studies have focused on theimmunogenicity and/or the protective efficacy of the resulting vaccinesand have not addressed vaccine associated pathology especially thoseoccurring after viral challenge.

In previous studies, while robust immune response and high levels ofprotection were observed using different MERS-CoV vaccine platforms,lung pathology was also noticed in vaccinated and challenged animals.These studies involved a variety of different MERS-CoV vaccinesincluding high dosage DNA-based vaccines, RBD or modified vaccinia virusankara (MVA) vaccines, or recombinant measles virus encoding MERS-CoV Sprotein vaccine; Muthumani et al.; Tai, W. et al. A recombinantreceptor-binding domain of MERS-CoV in trimeric form protects humandipeptidyl peptidase 4 (hDPP4) transgenic mice from MERS-CoV infection.Virology. 499, 375-382 (2016); Volz, A. et al. Protective Efficacy ofRecombinant Modified Vaccinia Virus Ankara Delivering Middle EastRespiratory Syndrome Coronavirus Spike Glycoprotein. J. Virol. 89,8651-8656 (2015); and Malczyk, A. H. et al. A Highly Immunogenic andProtective Middle East Respiratory Syndrome Coronavirus Vaccine Based ona Recombinant Measles Virus Vaccine Platform. J. Virol. 89, 11654-1167(2015); Agrawal et al.; Weingartl et al.; Tseng et al.; As apparent fromthese prior studies, a way to protect against MERS-CoV while safelyavoiding inducing severe lung pathology following immunization has yetto be developed.

In view of the limitations and problems with MERS-CoV vaccines, theinventor sought to develop a safe and efficacious vaccine for MERs-CoVthat would safely provide durable, broad, potent and universalprotection against MERS-CoV.

SUMMARY OF THE INVENTION

The invention involves an immunogenic CD40-targeted trimeric MERS-CoV S1fusion polypeptide as well as a corresponding polynucleotide encoding itand its use for safely inducing immune responses directed againstMERS-CoV without inducing vaccine associated respiratory pathologiesassociated with non-targeted vaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Schematic representation of MERS-CoV spike protein with both S1and S2 subunits. N refers to amino-terminus and C refers to thecarboxyl-terminus.

FIG. 1B. Schematic representation of MERS-CoV spike protein S1 subunitand N terminal signal peptide with no S2 subunit.

FIG. 2. Schematic representation of a chimeric nucleic acid encoding aprototype vaccine rAd5-S/Ag/F/CD40L. P: promoter such as a CMV promoter,S: leader sequence, Ag: coding sequence of antigen of interest, tag:affinity tag sequence for protein purification such as histidine tag, F:T4 bacteriophage fibritin trimerization motif sequence, L: Linkersequence (e.g., nonpolar amino acids), CD40L: ectodomain of CD40 ligand(CD40L), Poly A: polyadenylation tail.

FIG. 3. The S1/F/CD40L fusion protein (SEQ ID NO: 11). Segments of thefusion protein from top to bottom: signal peptide, MERS-CoV S1,Histidine tag for affinity purification, bacteriophage T4 fibritintrimerization motif, nonpolar amino acid linker (AAA) and an ectodomainof CD40L.

FIG. 4A. Schematic representation of DNA construct expressingCD40-targeted MERS-CoV vaccine rAd5-S1/F/CD40L which was engineered toexpress a fusion protein with the S1 subunit of the MERS-CoV S proteinwhich contains the N-terminal S protein signal peptide, followed by atrimerization motif derived from T4 bacteriophage fibritin (F) fusedwith the ectodomain of mouse CD40 ligand at the C-terminus (CD40L). TheS1 and CD40L are expressed within a trimerized fusion protein via F(S1/F/CD40L). The construct was placed under the control of a CMVpromoter (CMV) and in front of BGH-poly A site (Poly A).

FIG. 4B. Schematic representation of a DNA construct expressing thenon-CD40 targeted MERS-CoV vaccine construct rAd5-S1.

FIG. 4C. Schematic representation of a control DNA construct expressingGFP instead of MERS-CoV antigenic determinants.

FIG. 5. Schematic representation of animal study design.

FIG. 6A MERS-CoV Spike rAd vaccine induced neutralizing antibodies(nAbs) after priming. MNT₅₀ denotes complete protection of 50% of Verocells in a well.

FIG. 6B MERS-CoV Spike rAd vaccine induced nAbs after boost.Neutralization titers for FIGS. 6A and 6B were determined as the highestserum dilutions from each individual mouse that completely protectedVero E6 cells in at least 50% of the wells (MNT₅₀). Titers are shown asmeans from 15 mice per group±s.d from one experiment. ***P<0.001,**P<0.01 and *P<0.05 (one-way ANOVA with Bonferroni's post-test).

FIG. 7A. Percent survival of mice after immunization and subsequentchallenge with MERS-CoV. Immunization with S1-based rAd vaccine rAd5-S1and S1-based CD40-targeted rAd5-S1/F/CD40L provided complete protectionagainst lethal MERS-CoV challenge, while all control animals (rAd-5-GFP)died. Groups of hDPP4 Tg+ mice were immunized with two doses of 10⁹ pfuof the indicated rAd constructs and challenged with 100 LD₅₀ of MERS-CoV(EMC2012 strain) 5 weeks post secondary immunization. Data are shownfrom one experiment with n=11 mice per treatment group.

FIG. 7B. Percentage body weight loss of two immunized groups compared tocontrol group (rAd5-GFP) described in FIG. 7A.

FIGS. 8A-8F. Histological comparison of lung pathology on day threepost-challenge after immunization with rAd5-GFP (control, FIGS. 8A and8D), non-targeted rAd5-S1 (FIGS. 8B and 8E) and CD40-targetedrAd5-S1/F/CD40L (FIGS. 8C and 8F) and challenge with MERS-CoV.Histopathologic evaluation was performed on deparaffinized sectionsstained by routine hematoxylin-eosin (H&E) staining. Arrows indicatemonocytic and lymphocytic infiltrates as well as perivascularhemorrhage.

FIG. 9. Pulmonary viral load was determined in the lungs of immunizedmice subsequently challenged with MERS-CoV. Viral load was determined asTCID50 equivalents (TCID eq/g) by qRT-PCR using a standard curvegenerated from dilutions of RNA from a MERS-CoV with known virus titer.Titers are shown as means from 4 mice per group±s.d from one experiment.***P<0.001 (one-way ANOVA with Bonferroni's post-test). Highlysignificant reductions in viral titer compared to control (rAd5-GFP)were found for rAd5-S1 and rAd5-S1/F/CD40L.

DETAILED DESCRIPTION

Toward an objective of providing a safe and effective vaccine toMERS-CoV, the inventor engineered and tested a vaccine platform thattargets MERS-CoV S1 antigen to CD40 expressed by antigen presentingcells. A vaccine containing the CD40 targeted S1 antigen(rAd5-S1/F/CD40L) was compared to an S1-based vaccine that did nottarget CD40 (rAd5-S1) and to a control vaccine without S1 (rAd5-GFP).Immunization of human DPP4 transgenic mice showed that a single dose ofthe targeted rAd5-S1/F/CD40L vaccine elicited highly significant levelsof neutralizing antibodies compared to that produced by thenon-CD40-targeted rAd5-S1 vaccine which required two doses to inducerobust response. As shown by MERS-CoV challenge, both vaccines conferredcomplete protection against infection and resulted in undetectableinfectious virus titers as well as significantly lower levels of viralRNA in the lungs as compared to the control. However, surprisingly, themice immunized with the non-targeted S1 vaccine (rAd5-S1), but not thoseimmunized with the CD40-targeted vaccine (rAd5-S1/F/CD40L), exhibitedpulmonary perivascular hemorrhage three days post challenge despite theobserved protection showing that immunization with S1 without CD40Ltargeting leads to vaccine-associated lung pathology after viralexposure.

These data indicate that using CD40L as a targeting molecule andmolecular adjuvant not only enhances immunogenicity and protectiveefficacy against MERS-CoV but also prevents such pulmonary pathology.Moreover, the CD40-targeted vaccine was more potent suggesting that alower dosage could be used further reducing the risk immunologicalside-effects. These results also show that immunization with a consensusS1 antigen sequence induces protective immune responses againstMERS-CoV.

MERS-CoV antigens include viral antigens encoded by the structuralprotein genes Spike (S), Envelope (E), Membrane (M) and nucleopcapside(N). MERS-CoV also expresses a polymerase. Spike (S) protein isassembled into trimers which form peplomers on the surface of the viralparticle that give the Coronaviridae family its name. Typically, animmunogen or vaccine containing a CD40-targeted polypeptide (apolypeptide that is directed or targeted to CD40 on antigen presentingcells) of the invention will only contain viral S protein, or only S1protein epitopes, for example, it will omit epitopes from other MERS-CoVantigens and a S1-specific immunogen will omit S2 epitopes. However, insome embodiments, such a vaccine may substitute or include one or moreother antigens or epitopes of non-S1 MERS-CoV antigens either as part ofa CD40-targeted polypeptide or as a separate ingredient of animmunogenic composition or vaccine.

Coronavirus antigens. In addition to MERS-CoV, five other types of humancoronaviruses are known. These are 229E (alpha coronavirus), NL63 (alphacoronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus) andSARS-CoV (the beta coronavirus that causes severe acute respiratorysyndrome, or SARS). Viral proteins involved in recognition, attachmentand invasion of human host cells from these other coronaviruses, such ascoronavirus S proteins, may be substituted for the S or S1 protein ofMERS-CoV in the polypeptide according to the invention. In combinationwith a CD40 ligand, these polypeptides may provide substantial immunityagainst coronaviruses and reduce the severity of side-effects associatedwith vaccination, such as vaccine-induced inflammation or immunologicalhypersensitivity to an exogenous antigen.

Animal coronaviruses include Infectious bronchitis virus (IBV) whichcauses avian infectious bronchitis; Porcine coronavirus (transmissiblegastroenteritis coronavirus of pigs, TGEV); Bovine coronavirus (BCV),responsible for severe profuse enteritis in of young calves; Felinecoronavirus (FCoV) causes mild enteritis in cats as well as severeFeline infectious peritonitis (other variants of the same virus); twotypes of canine coronavirus (CCoV) (one causing enteritis, the otherfound in respiratory diseases); Turkey coronavirus (TCV) causesenteritis in turkeys; Ferret enteric coronavirus causes epizooticcatarrhal enteritis in ferrets; Ferret systemic coronavirus causesHP-like systemic syndrome in ferrets; Pantropic canine coronavirus;porcine epidemic diarrhea virus (PED or PEDV), has emerged around theworld. Its economic importance is as yet unclear, but shows highmortality in piglets. In some embodiments, the invention is directed toimmunogenic polypeptides containing a ligand targeting CD40 and an S1protein analog from another coronavirus which replaces the MERS-CoV S1determinants in a CD40-targeted MERS-CoV S1 fusion proteins.

The platform disclosed herein can be used for other viruses instead ofMERS-CoV. It could be used with any other viral antigen or fragmentthereof, or variant thereof including but not limited to a virus fromone of the following families: Adenoviridae, Arenaviridae, Bunyaviridae,Caliciviridae, Coronaviridae, Hepadnaviridae, Herpesviridae,Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae,Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, orTogaviridae. The viral antigen can be from human papillomoa virus (HPV),human immunodeficiency virus (HIV), polio virus, hepatitis B virus,hepatitis C virus, smallpox virus (Variola major and minor), vacciniavirus, influenza virus, rhinoviruses, dengue fever virus, equineencephalitis viruses, rubella virus, yellow fever virus, Norwalk virus,hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cellleukemia virus (HTLV-II), California encephalitis virus, Hanta virus(hemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus,measles virus, mumps virus, respiratory syncytial virus (RSV), herpessimplex 1, herpes simplex 2, varicella-zoster virus, cytomegalovirus(CMV), Epstein-Barr virus (EBV), flavivirus, foot and mouth diseasevirus, chikungunya virus, lassa virus, arenavirus, Nipah virus, Lassavirus or cancer causing virus.

Middle East respiratory syndrome coronavirus. Various strains ofMERS-CoV viruses have been isolated. Isolates of MERS CoV are describedby accession KF186567.1 and KT026454.1 which are incorporated byreference. Another commonly used strain is described by accession numberJX869059.2 (Human betacoronavirus 2c EMC/2012) which is alsoincorporated by reference.

Some embodiments of the invention involve the use of polynucleotides orproteins encoded by polynucleotides of different isolates of MERS CoV orvariants having 95, 96, 97, 98, 99, 99.5, 99.9, <100 or 100% sequenceidentity to the isolates identified by accession/version numberKF186567.1, JX869059.2 or KT026454.1.

MERS S protein is a structural spike protein encoded by the MERS CoVgenome. The S glycoprotein consists of a globular S1 domain at theN-terminal region (e.g., residues 1-725), followed by membrane-proximalS2 domain, a transmembrane domain and an intracellular domain as shownin FIG. 1A. Sequences of some kinds of S proteins are described byaccession/version numbers AGN70962.1 and AKK52592.1.

Natural or synthetic variants of S protein and its S1 and S2 subunitsare also encompassed by the invention. These include S, S1 or S2 proteinvariants that have amino acid sequences that are at least 98, 99, 99.5,99.9, <100% identical or similar to the amino acid sequences describedby the above accession/version numbers or to the consensus S1 amino acidsequence of SEQ ID NO: 2, which may be encoded by the polynucleotidesequence of SEQ ID NO: 1. Immunogenic fragments of S proteins are alsoincluded as well as receptor binding portions of the S1 proteinincluding those described by FIG. 1B.

CD40 is a costimulatory protein found on antigen presenting cells and isrequired for their activation. In the macrophage, the primary signal forCD40 activation is IFN-γ from Th1 type CD4 T cells. The secondary signalis CD40L (CD154) on the T cell which binds CD40 on the macrophage cellsurface. As a result, the macrophage expresses more CD40 and TNFreceptors on its surface increasing its level of activation.

Activation results in the induction of potent microbicidal substances inthe macrophage including reactive oxygen species and nitric oxideleading to the destruction of ingested microbes but which could alsoinduce immunological side-effects. Surprisingly, the inventor found thatCD40-based targeting of S1 protein to antigen presenting cells reducedvaccine-associated side-effects despite contact of CD40 on antigenpresenting cells with a CD40 ligand.

Human CD40 reference nucleic acid sequences include NM_001250 (SEQ IDNO: 18), NM_001302753, NM_152854, NM_001322421, and NM_001322422, eachof which is incorporated by reference along with the correspondingencoded polypeptides. For example, SEQ ID NO: 18 encodes the amino acidsequence for human CD40 described by SEQ ID NO: 19. Vaccines foradministration to humans will generally encompass a CD40L or a CD40Lvariant that bind to human CD40, or a CD40L ligand or variant that bindsto the CD40 of a non-human animal being vaccinated.

Camelid CD40 encoding polynucleotides are described by NCBI ReferenceSequences: XM_010978627.1 and XM_010978624.1 (SEQ ID NO: 20). A camelidCD40 amino acid sequence is described by SEQ ID NO: 21. Vaccines foradministration to camelids will generally encompass a CD40L or a CD40Lvariant that binds to a camelid CD40.

Additional CD40 sequence isoforms or homologs are known and publicallyavailable via PubMed or in other public polynucleotide or polypeptidedatabases such as at: hypertext transfer protocolsecure://worldwideweb.ncbi.nlm.nih.gov/protein/?term=cd40 or to(hypertext transferprotocol://worldwideweb.ncbi.nlm.nih.gov/nuccore/?term=cd40 (both lastaccessed Aug. 27, 2018 and both incorporated by reference). Vaccines foradministration to other animals besides humans and camelids willgenerally encompass a CD40L or a CD40L variant that bind to acorresponding CD40 molecule from the relevant species.

CD40L (CD40 Ligand, CD154) is a protein that is primarily expressed onactivated T cells and is a member of the TNF superfamily of molecules.It binds to CD40 on antigen-presenting cells (APC), which leads to manyeffects depending on the target cell type. CD40L has three bindingpartners: CD40, α5β1 integrin and αIIbβ3. CD40L is a type II membraneprotein which exists as either cell-surface protein or secreted protein.The cell surface-attached protein consists of cytoplasmic domain (CD) atits N-terminus, transmembrane domain (TMD) and an extracellular domain(ECD) at its C-terminus. On the other hand, the soluble form is mainlythe extracellular domains of CD40L missing both CD and TMD. As shown inthe Example, the inventor used a coding region of the soluble form/ECDas it is responsible for CD40 binding on APCs by deleting the CD and TMDcoding regions.

A variety of different CD40L polynucleotide and polypeptide sequencesare known from different animals including humans (NM_000074.2,NP000065.1, GenBank: BC071754.1, SEQ ID NO: 22), Arabian camels(XP_010993166.1, XM_010994864.1, SEQ ID NO: 24), Bactrian camels(XP010950821.1), wild Bactrian camels (XP006195602.1; NCBI ReferenceSequence: XM_010952519.1; SEQ ID NO: 26), horses (AEB61022.1;XP005614588.1), cattle (XP024844321.4 bison (XP010851595.1), sheep (NP001068569.1), goats (XP005700481.1), dogs (NP001002981.1), cats(NP001009298.1) and mice (NP_035746.2). Additional CD40L sequencehomologs are known and publically available via PubMed or in otherpublic polynucleotide or polypeptide databases such as at: (hypertexttransfer protocolsecure://worldwideweb.ncbi.nlm.nih.gov/protein/?term=cd40+ligand or to(hypertext transfer protocolsecure://worldwideweb.ncbi.nlm.nih.gov/nuccore/?term=cd40+ligand (bothlast accessed Aug. 27, 2018 and both incorporated by reference). In someembodiments of the invention, a CD40L from a human or non-human animalwill be incorporated into a fusion polypeptide as described herein. Insome embodiments a variant CD40L sequence may be used. Variant CD40Lamino acid sequences that bind to CD40 may be at least 70, 75, 80, 85,90, 95, 96, 97, 98, 99, <100% identical or similar to the CD40Lsequences disclosed herein.

Trimerization motif. The CD40-targeted S1 polypeptide of the inventionincludes a trimerization motif which facilitates or enables formation ofa trimer of the monomeric CD40-targeted polypeptides. One suchtrimerization motif is described by a foldon sequence derived from T4phage fibritin described in SEQ ID NO: 6 and encoded by thepolynucleotide of SEQ ID NO: 5. This sequence when incorporated into apolypeptide can stabilize formation of a triple helix by the componentprotein monomers. Other trimerization motifs include those described by,and incorporated by reference to Kammerer, et al., PNAS 102(39):13891-13896 or variants of the T4 phage fibritin sequence of SEQ ID NO:6 which contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions,substitutions, or insertions and which retain an ability to formtrimers. CD40L and MERS-CoV S protein are both trimers. A trimerizationmotif may be used to connect the surface exposed region from MERS-CoVspike protein (i.e. S1) to the soluble from of CD40L. The inventor foundthat trimerization leads to targeting of S1 to CD40-expressing cells,increasing binding avidity to CD40 receptor, promoting APCs (such as DCsand macrophages) activation and maturation, and stabilization of nativetrimeric conformation of both S1 and CD40L, a property necessary forefficient CD40 binding and function. As shown herein, a short fibritinfragment (27 aa) may be sufficient for forming stable trimers of abiologically active recombinant fusion protein. In one alternativeembodiment, a trimerization motif that facilitates trimerization of S orS1 and/or CD40L may be, respectively, omitted as monomeric or dimericforms of S1 can exhibit immunological activity and monomeric or dimericforms of CD40L can bind to CD40.

Linkers may be used next to one or more elements of the invention,including next to S1 protein sequences, tags (e.g., His tag), or CD40ligand sequences to facilitate the functions of these elements, such asto enhance the ability of a His-tag to bind to a substrate or enhancethe ability of CD40 ligand to bind to CD40 on an antigen presentingcell. Flexible, rigid, or cleavable linkers may be used. Fusion proteinlinkers are described by, and incorporated by reference to Chen, X., etal., Fusion protein linkers: Property, design and functionality, Adv.Drug Deliv. Rev. 65(10):1357-1369. One such linker is the non-polaramino acid linker described by SEQ ID NO: 8 and encoded by SEQ ID NO: 7,though other non-polar linkers may also be used. A linker may be asingle amino acid residue or contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues, preferablyalanine or other non-polar amino acid residues.

A signal peptide sequence may be incorporated into a CD40-targeted S1fusion polypeptide at its N-terminus to traffic the polypeptide out of acell in which it is expressed. Signal peptides are extremelyheterogeneous and many prokaryotic and eukaryotic signal peptides arefunctionally interchangeable even between different species. Theefficiency of protein secretion is determined by the signal peptide, seeKober L, Zehe C, Bode J (April 2013). “Optimized signal peptides for thedevelopment of high expressing CHO cell lines”. Biotechnol. Bioeng. 110(4): 1164-73. doi:10.1002/bit.24776. PMID 23124363; von Heijne G (July1985). “Signal sequences: The limits of variation”. J Mol Biol. 184 (1):99-105. doi: 10.1016/0022-2836(85)90046-4. PMID 4032478 bothincorporated by reference.

As shown in the Example, a CD40-targeted S1 fusion protein wasengineered to be secretable by deleting the cytoplasmic andtransmembrane domains from both S protein and CD40L and by the additionof the human tyrosinase signal peptide. However, as shown below nosignal peptide was maintained from S protein, but human tyrosinasesignal peptide or IL2 signal sequence can used as well as longer orshorter signal peptide sequences. The presence of the signal peptide(secretory signal sequence) upstream of the fusion protein should renderthe fusion protein secretable from in vivo infected cells upon. Geneproducts with a secretion signal produced by adenovirus-infected cellswould be secreted to increase the antigen load and expression in vivo.The secretory signal sequence directs the S1-CD40L fusion protein tocompartments of the cell which cause the fusion protein to be secretedfrom the cell. The same is expected if using DNA vaccine or otherdelivery vectors. Once the fusion protein is secreted it will betargeted to CD40-expressing cells (APCs) to enhance the immune response.

A signal peptide, such as residues 1-18 of SEQ ID NO: 12, is used insome embodiments of the invention.

In other embodiments, one skilled in the art may select an appropriatesignal peptide for use with the invention. Suitable signal proteinsequences are known in the art for various kinds of host cells. Forexample, a signal peptide encoded by one of the vectors available in acommercial kit may be used, see for example, from the Mammalian SignalPeptide Vector Set; hypertext transfer protocolsecure://worldwideweb.sigmaaldrich.com/catalog/product/sigma/pp2379?lang=en&region=US(incorporated by reference, last accessed Aug. 24, 2018).

In other embodiments, where it is not desired to secrete a CD40-targetedS1 fusion polypeptide, a signal peptide may be omitted and thepolypeptide recovered by lysis of the host cell. Other mammalianexpression sequences or vectors such as those described by hypertexttransfer protocolsecure://worldwideweb.sigmaaldrich.com/life-science/molecular-biology/cloning-and-expression/vector-systems/transient-expression.html(last accessed Aug. 24, 2018, incorporated by reference) may also beused.

Protein tags. In one embodiment, the CD40-targeted polypeptide of theinvention comprises a His-tag (SEQ ID NO: 4) which can be encoded by thepolynucleotide sequence of SEQ ID NO: 3. Such tags are known in the artand incorporated by reference to hypertext transfer protocolsecure://en.wikipedia.org/wiki/Polyhistidine-tag (last accessed Aug. 24,2018). This stretch of histidine residues incorporated into theCD40-targeted polypeptide permits it to be recovered by affinitypurification, for example, by binding between histidine and a metal ionsuch as copper, nickel, zinc, or cobalt. As the metal ion, copper hasthe highest affinity, and the affinity decreases in the order of nickel,zinc, and cobalt. Nickel is often used for ordinary purposes, and cobaltis used when it is desired to increase the degree of purification.Affinity purification using a polyhistidine-tag usually results inrelatively pure protein when the recombinant protein is expressed inprokaryotic organisms. For example, prokaryotic or eukaryotic cellsexpressing a CD40-targeted polypeptide can be harvested viacentrifugation and the resulting cell pellet lysed either by physicalmeans (e.g., shearing, french-press, sonoication) or by means ofdetergents and enzymes such as lysozyme or any combination of these toproduce a raw lysate containing the recombinant CD40-targeted proteinamong many other proteins originating from the host cell. This mixtureis incubated with an affinity resin containing bound divalent nickel orcobalt ions, which are available commercially in different varieties.Nickel and cobalt have similar properties and as they are adjacentperiod 4 transition metals (v. iron triad). These resins are generallysepharose/agarose functionalised with a chelator, such as iminodiaceticacid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel andcarboxylmethylaspartate (Co-CMA) for cobalt, which the polyhistidine-tagbinds with micromolar affinity. The resin is then washed with phosphatebuffer to remove proteins that do not specifically interact with thecobalt or nickel ion. With Ni-based methods, washing efficiency can beimproved by the addition of 20 mM imidazole (proteins are usually elutedwith 150-300 mM imidazole). Generally nickel-based resins have higherbinding capacity, while cobalt-based resins offer the highest purity.The purity and amount of protein can be assessed by SDS-PAGE and Westernblotting.

Other kinds of protein tags, such as those described by SEQ ID NOS:13-17 may also be used to purify a fusion protein of the invention bybinding to their corresponding substrates.

In some embodiments a polynucleotide sequence encoding a his-tag orother tag will be positioned after the start codon for the CD40-targetedpolypeptide, in others, it may be positioned in the body of thepolypeptide, for example, between residues derived from S1 and thosederived from a CD40-binding ligand, and in others it may be positionedat the C-terminal of the polypeptide.

While tags are often incorporated at a C- or N-terminal of a protein aHis tag of the invention as shown in the Example was placed the body ofthe fusion protein to facilitate recovery of the trimerized protein. Theinventors found that this placement avoids cleavage of an N-terminallyplaced tag along with the signal peptide which usually occurs duringprotein secretion. Additionally, placing the tag in the body of thefusion protein can increase the flexibility of the fusion proteinallowing it to more easily form a trimer.

In some embodiments, a polynucleotide sequence encoding a linker (e.g.,gly-gly-gly or gly-ser-gly, or other linkers described herein) may beplaced so as to encode an amino acid linker between residues derivedfrom S1 protein or those derived from a CD40-binding ligand and the tag.Such a linker may be selected and positioned so as to prevent the tagfrom affecting the biological activity of the CD40-targeted polypeptide(e.g., recognition by the immune system or ability to bind to CD40) orto facilitate binding of the tag to its binding partner during affinitypurification.

Any tag that can be captured by a binding molecule immobilized on asolid matrix can be used. For example, a poly-His-tag may be replaced byanother affinity purification ligand such as a tag containingalternative histidine/glutamine or histidine/asparagine residues; e.g.,tags comprising HQHQHQ (SEQ ID NO: 13) or (SEQ ID NO: 14) or a HAT tagsuch as KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 15) which like poly-His tagsalso bind to metals described above via histidine. Other protein tagssuch as (strep)avidin, which binds to biotin, or Strep-tag II which issynthetic peptide consisting of eight amino acids,Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:17) which bindsStrep-Tactin®, a specifically engineered streptavidin. Other proteintags and their corresponding binding ligands for affinity purificationare known in the art and may be used instead of a His-tag. These includeFLAG-tag, which comprises DYKDDDDK (SEQ ID NO: 16), chitin bindingprotein (CBP), maltose binding protein (MBP) andglutathione-S-transferase (GST).

In some embodiments, the tag will be removed during purification of theCD40-targeted polypeptide or after its trimerization, in others it maybe left in the CD40-targeted polypeptide. In the embodiment exemplifiedherein, the His-tag is retained in the final vaccine fusion proteinproduct, however, in other embodiments the fusion protein tag may beengineered to be removed from the final protein product.

The various segments of the CD40-targeted S1 fusion protein of theinvention may appear in the order shown by FIG. 2 or 3 or in alternateorders that do not disrupt trimer formation or CD40 binding, forexample, the CD40 ligand segment may appear toward the N-terminal of thefusion polypeptide and the S1 segment toward the C-terminal, or the tagmay appear at the N- or C-terminal.

The polynucleotide and polypeptide sequences disclosed herein, such asthose for MERS-CoV S1 protein, CD40 or CD40-like proteins, trimerizationmotifs, linkers, tags, and signal peptides, include those havingsequence identity or similarity to the disclosed sequences, for example,that have between 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, <100, or and100% sequence identity or similarity to the disclosed polynucleotides orpolypeptides. Typically, these variant proteins having substantialsequence identity or similarity will encode, or comprise, polypeptidesthat exhibit the same or similar properties as the disclosed sequence,for example, a variant S1 protein will exhibit similar immunologicalproperties to a disclosed S1 sequence, a variant trimerization motifwill facilitate trimerization, and a variant CD40-ligand will bind toCD40.

BLASTN may be used to identify a polynucleotide sequence having at least70, 75, 80, 85, 90, 95, 96, 97, 98, 99, <100, or and 100% (or anyintermediate %) sequence identity to a reference polynucleotide. Arepresentative BLASTN setting optimized to find highly similar sequencesuses an Expect Threshold of 10 and a Wordsize of 28, max matches inquery range of 0, match/mismatch scores of 1/−2, and linear gap cost.Low complexity regions may be filtered/masked. Default settings aredescribed by and incorporated by reference to to hypertext transferprotocolsecure://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome(last accessed Aug. 7, 2018).

BLASTP can be used to identify an amino acid sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96, 97.5%, 98%, 99%, <100%or 100% (or any intermediate %) sequence identity or similarity to areference amino acid using a similarity matrix such as BLOSUM45,BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely relatedsequences, BLOSUM62 for midrange sequences, and BLOSUM80 for moredistantly related sequences. Unless otherwise indicated a similarityscore will be based on use of BLOSUM62. When BLASTP is used, the percentsimilarity is based on the BLASTP positives score and the percentsequence identity is based on the BLASTP identities score. BLASTP“Identities” shows the number and fraction of total residues in the highscoring sequence pairs which are identical; and BLASTP “Positives” showsthe number and fraction of residues for which the alignment scores havepositive values and which are similar to each other. Amino acidsequences having these degrees of identity or similarity or anyintermediate degree of identity or similarity to the amino acidsequences disclosed herein are contemplated and encompassed by thisdisclosure. A representative BLASTP setting that uses an ExpectThreshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and GapPenalty of 11 (Existence) and 1 (Extension) and a conditionalcompositional score matrix adjustment. Default settings for BLASTP aredescribed by and incorporated by reference to to hypertext transferprotocolsecure://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome(last accessed Aug. 7, 2018).

Vaccine platforms. A number of different vaccine platforms can be usedto expose a subject's immune system to the CD40-targeted MERS-CoV Sprotein vaccine of the invention. These include a subunit vaccine from apurified trimeric fusion protein disclosed herein or from animmunogenically active fragment of the trimer; a DNA or RNA subunitvaccine prepared from DNA or RNA, or modified DNA or RNA, encoding thefusion protein, for example, for intramuscular injection of a plasmid orconstruct carrying a DNA or RNA subunit vaccine that encodes a fusionprotein according to the invention that when injected expresses thefusion protein in the vaccinated subject; or a killed, nonreplicating,attenuated or living viral vaccine that carries a CD40-targeted fusionprotein according to the invention or carries DNA or RNA encoding aCD40-targeted fusion protein, A virus-based vaccine may use one of manyavailable virus platforms including an adenovirus 5 platform or rADSvector; see e.g. Amalfitano A, Hauser M A, Hu H, Serra D, Begy C R,Chamberlain J S. Production and characterization of improved adenovirusvectors with the E1, E2b, and E3 genes deleted. J Virol. 1998;72:926-933; Gabitzsch, et al., Vaccine. 2009 Oct. 30; 27(46): 6394-6398(both incorporated by reference). Other viral vectors that accommodate achimeric gene that encodes CD40-targeted fusion protein according to theinvention may also be selected.

In some embodiments a vaccine may be designed as a DIVA vaccine which(Differentiating Infected from Vaccinated Animals) which carry at leastone epitope less than MERS-CoV strains circulating in the field. Such avaccine facilitates later serological differentiation between infectedand vaccinated animals by screening for the missing epitope, forexample, with a monoclonal antibody that recognizes it in infectedanimals, but not in vaccinated, not infected animals.

A vaccine formulation, whether a protein-based vaccine, nucleicacid-based vaccine or virus-based vaccine may include additionalingredients such as excipients or adjuvants.

DNA-based vaccines. A chimeric nucleic acid in an acceptable liquid maybe utilized as a direct immunizing agent, for example, as generallydescribed by Felgner, et al., U.S. Pat. No. 5,589,466 or by Leitner, etal., Vaccine. 1999 Dec. 10; 18(9-10): 765-777, both of which areincorporated by reference. Vectors and procedures suitable for use inDNA vaccination are known and are also incorporated by reference tohypertext transfer protocol secure://en.wikipedia.org/wiki/DNAvaccination (last accessed Oct. 3, 2018) and the articles cited therein.

Vaccine vectors include but are not limited to the Ad5 vector describedby the inventors as well as vectors described by, and incorporated byreference to Choi, et al., Clin Exp Vaccine Res. 2013 July; 2(2):97-105. These include adenovirus, alphavirus and pox virus vectors.Those skilled in the art may incorporate nucleic acids encoding thechimeric polypeptides of the invention into such vectors and administersuch vectors according to current medical protocols.

Excipients. Typically, vaccines and/or immunogenic compositions of theinvention will contain a pharmaceutically acceptable carrier or diluent.Carriers include, but are not limited to, stabilizers, preservatives,and buffers. Suitable stabilizers are, for example SPGA, polysorbate(Tween®) compositions, carbohydrates (such as sorbitol, mannitol,starch, sucrose, dextran, glutamate, or glucose), or proteins (such asalbumin). Suitable preservatives include thimerosal, merthiolate, andgentamicin. Diluents include water and other aqueous buffers (such asnormal or buffered saline), alcohols, and polyols such as glycerol.Vaccines and/or immunogenic compositions according to the variousembodiments disclosed herein can be prepared, stored or distributed insolid, semisolid, liquid or aerosol form, for example, as alyophilizate, a frozen solution, emulsion or suspension, a liquidsolution, suspension or emulsion, or in a aerosol form.

Adjuvants. In some formulations of the vaccines and/or immunogeniccompositions, suitable excipients, stabilizers, and the like may beadded as are known by persons of ordinary skill in the art. In otherembodiments, the immunogenic compositions further include or areadministered with an adjuvant, such as an aluminum salt (e.g., aluminumhydroxide, aluminum phosphate, and aluminum potassium sulfate) ormonophosphoryl lipid A which have been safely used in U.S. vaccines. Inother formulations, no adjuvant will be incorporated with aCD40-targeted polypeptide of the invention.

Adjuvants suitable for use in animals include, but are not limited to,Freund's complete or incomplete adjuvants, Sigma Adjuvant System (SAS),and Ribi adjuvants. Adjuvants suitable for use in humans include, butare not limited to, MF59 (an oil-in-water emulsion adjuvant); MontanideISA 51 or 720 (a mineral oil-based or metabolizable oil-based adjuvant);aluminum hydroxide, -phosphate, or -oxide; HAVLOGEN®. (an acrylic acidpolymer-based adjuvant, Intervet Inc., Millsboro, Del.); polyacrylicacids; oil-in-water or water-in-oil emulsion based on, for example amineral oil, such as BAYOL or MARCOL™. (Esso Imperial Oil Limited,Canada), or a vegetable oil such as vitamin E acetate; and saponins.Components with adjuvant activity are widely known and, generally, anyadjuvant may be utilized that does not adversely interfere with theefficacy or safety of the vaccine and/or immunogenic composition.

Method of inducing immunity. The invention contemplates a method forinducing an immune response in a subject or vaccinating a subject. Thisimmune response includes at least one of a cellular or humoral response,or both, to MERS-CoV induced by the disclosed CD40-targeted fusionproteins. As shown by the inventor, the immune responses induced by aCD40-targeted S1 polypeptide are qualitatively different than thoseinduced by a non-targeted S1 polypeptide in that the non-targeted S1polypeptide induces vaccine-associated side-effects as shown by FIGS.8A-8F.

The immunogenic CD40-targeted polypeptide or DNA encoding it can beformulated in accordance with standard techniques well known to thoseskilled in the pharmaceutical art and can be administered in dosages andby techniques well known to those skilled in the medical arts takinginto consideration such factors as the age, sex, weight, respiratorystatus, and condition of the particular subject, and the route ofadministration.

The immunogenic CD40-targeted polypeptide or DNA/RNA encoding istypically administered intradermally, subcutaneously, submucosally,intranasally, intrapulmonarily, intramuscularly or by other parenteralroute. Some other modes of administration include oral, administrationon to or through a mucous membrane, such as to ocular tissue, sinustissue, bronchial tissue, pulmonary tissue, enteric tissue, vaginaltissue, or rectal tissue.

An effective amount of the immunogenic CD40-targeted polypeptide orDNA/RNA encoding it is an amount that prevents or reduces the severityof or otherwise ameliorates MERS-CoV infection, reduces MERS-CoV titer,or alleviates the severity of symptoms of a MERS-CoV infection. Efficacymay be determined by comparing the level of protection in vitro or invivo after immunization with that before immunization or by comparisonto an unimmunized control group.

An immunogenic polypeptide or nucleic acid according to the inventionmay be administered prophylactically or therapeutically. In prophylacticadministration, the vaccines can be administered in an amount sufficientto induce an immune response that protects against subsequent exposureto MERS-CoV, reduces the risk of being infected, or that reduces theseverity of symptoms should a vaccinated subject become infected.Prophylactic administration of a vector, carrier or reservoir ofMERS-CoV can reduce the risk of transmission to other susceptiblesubjects or eliminate or reduce viral titers in a carrier or reservoirof MERS-CoV. Prophylactic administration can also reduce the risk oftransmission from food products, such as milk or meat, obtained from ananimal that may be a MERS-CoV carrier or reservoir, for example, it mayboost anti-MERS-CoV antibody titers in camelid milk, rendering it moresafe for human consumption.

In therapeutic applications, the vaccines are administered to a subjectin need thereof in an amount sufficient to elicit a therapeutic effect.An amount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend on, e.g.,the particular composition of the vaccine regimen administered, themanner of administration, the stage and severity of the disease, thegeneral state of health of the patient, and the judgment of theprescribing physician. Preferably, an effective amount is selected thatdoes not induce significant side-effects.

Vaccine administration. The vaccines and/or immunogenic compositions ofthe invention can be administered by immunization methods known to thoseof skill in the art. These include a single immunization or multipleimmunizations in a prime-boost strategy. A boosting immunization can beadministered at a time after the initial immunization that is days,weeks, months, or even years after the prime immunization. A boostimmunization may be administered 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 16, 20, or 24 months after the initial priming immunization.

In vitro or ex vivo immunization. In some embodiments, antigenpresenting cells of a subject that express CD40 may be contacted with animmunogenic composition of the invention, then reinfused or otherwisereturned to a subject. CD40 is constitutively expressed by antigenpresenting cells, including dendritic cells, B cells and macrophages.For example, buffy coat cells may be isolated from the blood of asubject and contacted with a CD40-targeted S1 polypeptide of theinvention for a time sufficient for binding of the CD40-targetedpolypeptide to the antigen presenting cells, optionally washed to removeunbound polypeptide, and reinfused or otherwise returned to a subject.Alternatively bone marrow, cord blood cells, or isolated or cultureddendritic cells or other APCs may be used as a source of, or to produce,antigen presenting cells. As shown by the inventions, targeting an S1polypeptide to antigen presenting cells using a ligand that binds toCD40 reduces vaccine-associated lung pathology compared to an untargetedS1 vaccine. In vitro or ex vivo immunization further reduces thenon-targeted exposure of a subject to S1 epitopes and reduces the riskof immunological or inflammatory side-effects.

Subjects. The invention is typically directed to treatment of subjectssusceptible to MERS CoV infection or subjects who are vectors, carriers,or reservoirs for this virus. Subjects include humans, as well as otherprimates, domestic animals, pets, such as camels or dromedaries, as wellas wild animals which may act as reservoirs or vectors of the virus suchas bats or birds. For non-human subjects a variant vaccine may beadministered that contains a homolog of human CD40 ligand or a similarimmune system protein.

DNA or RNA based vaccines. In some embodiments, DNA encoding aCD40-targeted S1 polypeptide of the invention may be replicated orsynthesized by known methods. The DNA is then formulated foradministration to a subject, for example, by intravenous, subcutaneous,intramuscular, intrapulmonary, or intralymphatic administration.DNA-based vaccines and methods of their use are known and areincorporated by reference to Tregoning, J S, et al., Using Plasmids asDNA Vaccines for Infectious Diseases. Microbiol Spectr. 2014 December;2(6). doi: 10.1128/microbiolspec.PLAS-0028-2014; Ramirez, L A, et al.,Therapeutic and prophylactic DNA vaccines for HIV-1. Expert Opin BiolTher. 2013 April; 13(4):563-73. doi: 10.1517/14712598.2013.758709;Williams, J A, Improving DNA vaccine performance through vector design.Curr Gene Ther. 2014; 14(3):170-89. The above-cited references are eachincorporated by reference.

In some embodiments, mRNA encoding a CD40-targeted S1 polypeptide of theinvention may be produced by transcribing or otherwise producing an RNAmolecule corresponding to DNA encoding the CD40-targeted polypeptide byknown methods. The RNA is then formulated for administration to asubject, for example, by intravenous, subcutaneous, intramuscular,intrapulmonary, or intralymphatic administration. RNA-based vaccines andmethods of using them to induce immunity are described by andincorporated by reference to Hubaud, A., RNA vaccines: a noveltechnology to prevent and treat disease, to hypertext transfer protocolsecure://sitn.hms.harvard.edu/flash/2015/rna-vaccines-a-novel-technology-to-prevent-and-treat-disease/and to Pardi, N., et al., mRNA vaccines—a new era in vaccinology Nat RevDrug Discov. 2018 April; 17(4):261-279. doi: 10.1038/nrd.2017.243. Epub2018 Jan. 12.

Immune responses. Prophylactic or therapeutic immune responses inducedby the polypeptide or nucleic acid based compositions of the inventioncan include humoral immune responses, such as activation of B cells andinduction of IgA, IgG or IgM against MERS CoV, and cellular immuneresponses such as priming or expansion of T cells that recognize MERSCoV. Cellular immune responses are useful in protection against MERS-CoVvirus infection with CD4+ and CD8+ T cell responses being important.CD8+ immunity is of importance in killing virally infected cells.

Other, non-limiting embodiments of the invention include those describedbelow.

An engineered polypeptide that includes at least one immunogenicfragment, receptor-binding domain, or epitope of S1 polypeptide ofMiddle East respiratory syndrome coronavirus (MERS-CoV), at least onetrimerization motif, and at least one ligand for CD40. This engineeredCD40-targeted fusion polypeptide may include a native, consensussequence from two or more isolates, or otherwise engineered MERS-CoV S1protein sequence. MERS-CoV isolates include those from humans, camelidsand other animals susceptible to infection by MERS-CoV or which act ascarriers or reservoirs of MERS-CoV, and those described by the Centersfor Disease Control at to hypertext transfer protocolsecure://worldwideweb.nc.cdc.gov/eid/article/20/8/pdfs/14-0663.pdf (lastaccessed Aug. 22, 2018, incorporated by reference).

As shown in the Example, a fusion protein containing a MERS-CoV S1consensus sequence was used to induce broad immune response by usingconserved sequences which should cover any possible variation in viralsequences. All available MERS-CoV S sequences from GenBank databaseincluding isolates from all clades were used to design the consensussequence. Such sequence may offer protection against broad range ofMERS-CoV clades, including emerging strains.

This engineered polypeptide may contain an amino acid sequence that isat least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, <100 or 100% (or anyintermediate value within this range) identical to the MERS CoV S1consensus amino acid sequence of SEQ ID NO: 2 or is at least 70, 75, 80,85, 90, 95, 96, 97, 98, 99, <100 or 100% (or any intermediate valuewithin this range) identical to the engineered fusion polypeptidedescribed by SEQ ID NO: 12. It may contain at 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, or 50 (or any intermediate integervalue) amino acid residue deletions, substitutions or insertions to theamino acid sequences of SEQ ID NO: 2 or of SEQ ID NO: 12. In someembodiments, one or more conservative amino acid substitutions may bemade to the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 12 or tothe signal peptide, S1, trimerization motif, or CD40 Ligand segments ofthe engineered polypeptide or to the linker or protein tag segments whenpresent. The one or more conservative amino acid substitutions will bein the same class as the amino acid residue being substituted. Classesinclude aliphatic (glycine, alanine, valine, leucine, and isoleucine),hydroxyl- or sulfur/selenium-containing amino acids (serine, cysteine,selenocysteine, threonine, and methinone), cyclic (proline,hydroxyproline), aromatic (phenylalanine, tyrosine, tryptophan), basic(histidine, lysine, and arginine) and acid or acid-amides (aspartate,glutamate, asparagine and glutamine). The MERS-CoV S1 protein binds tothe DPP4 (CD26) host cell receptor. Preferably, the S1 component of theengineered polypeptide contains amino acid residues involved in bindingto this receptor.

A consensus sequence of S1 amino acid residues or other amino acidresidues of the engineered polypeptide, such as CD40 ligand residues,may be constructed based on analysis of an alignment of multiplesubtypes of a particular amino acid sequence, such as S1 amino acids ofdifferent kinds of MERS-CoV isolates. The consensus sequence may be usedto induce broad immunity against multiple subtypes, serotypes, orstrains of a particular viral antigen, such as the S1 subunit ofMERS-CoV. In some embodiments, the polypeptide may include an S1consensus sequence derived from the sequences of 2, 3, 4, 5, 6, 7, 8, 9,10 or more different MERS-CoV S1 amino acid sequences. A MERS-CoVconsensus sequence may be derived from the most virulent and/or mostprevalent MERS-CoV strains in a geographical area at a particular time.Tools for deriving consensus sequences are well known in the art andinclude JalView and UGENE; see Waterhouse, A. M., Procter, J. B.,Martin, D. M. A, Clamp, M. and Barton, G. J. (2009) Jalview Version 2—amultiple sequence alignment editor and analysis workbench,Bioinformatics 25 (9) 1189-1191 doi: 10.1093/bioinformatics/btp033;Okonechnikov K, Golosova O, Fursov M, the UGENE team. Unipro UGENE: aunified bioinformatics toolkit. Bioinformatics 2012 28: 1166-1167.doi:10.1093/bioinformatics/bts091 (both incorporated by reference).

An alternative to producing a MERS-CoV consensus sequence is to produceseparate engineered CD40-targeted fusion polypeptides that have S1 aminoacid sequences from 2, 3, 4, 5, 6, 7, 8, 9, or 10 different MERS-CoVisolates and then combine the engineered fusion polypeptides into amultivalent MERS-CoV vaccine.

Another alternative is to produce a single engineered CD40-targetedfusion polypeptide containing S1 epitopes from different MERS-CoVisolates. For example, the S1 receptor binding domains (or epitopesthereof) of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more MERS-CoV isolates may becombined into a hybrid S1 segment of an engineered polypeptide accordingto the invention to provide a multivalent response against differentMERS-CoV isolates.

This engineered CD40-targeted fusion polypeptide may also include anative, consensus sequence from two or more CD40 ligands, or otherwiseengineered CD40 ligand sequence, including fragments or variants of anative CD40 ligand sequence that bind to CD40. Human CD40L is describedby SEQ ID NO: 23 and camelid CD40Ls by SEQ ID NO: 25 and 27, thoughnaturally-occurring variants, such as transcript variants, whichgenerally have at least 95-<100% sequence identity to these sequences isalso included. Orthologs of the CD40Ls described herein from otherspecies that are at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, <100%identical or similar to the CD40L disclosed herein and that bind to CD40are also included.

Typically the CD40 ligand will be selected to recognize and bind to theCD40 molecule on antigen presenting cells of a subject to be vaccinated,for example, human CD40 for human subjects and camelid CD40 for camelidsubjects. Human CD40 is described by SEQ ID NO: 18 and camelid CD40 bySEQ ID NO: 20, though naturally-occurring variants, such as transcriptvariant, homologs, or paralogs, which generally have at least 95-<100%sequence identity to these sequences is also included. Variant CD40Lamino acid sequences that bind to CD40 may be at least 70, 75, 80, 85,90, 95, 96, 97, 98, 99, <100% identical or similar to the CD40Ldisclosed herein.

An engineered fusion CD40-targeted fusion polypeptide of the inventiontypically includes at least one trimerization motif which facilitates orenables trimerization of the polypeptide into a form similar to a nativetrimeric structure on the MERS-CoV surface. Trimerized forms of S1present both linear and conformation epitopes to which the humoral andcellular immune system can response. One such trimerization motif isdescribed by a foldon sequence derived from T4 phage fibritin describedin SEQ ID NO: 6 though other trimerization sequences may be used. Thissequence when incorporated into a polypeptide stabilizes formation of atriple helix by the component protein monomers.

An engineered CD40-targeted S1 fusion polypeptide of the invention maycontain additional elements to facilitate its expression orpurification. For example, it may contain a leader sequence that permitsits export from a host cell that expresses it, a linker that enhancesits immunological activity or ability to bind to CD40 on antigenpresenting cells or which sterically positions its component amino acidsequences, or a protein tag that facilitates its purification.

A signal peptide may be incorporated that facilitates expression andtrafficking of a polypeptide of the invention. One example of a signalpeptide is the native signal peptide of the MERS-CoV S protein or avariant thereof, though other signal peptides suitable for use inparticular host cells may be used instead, such as those described byKober, L, et al. (2013), id or by vonHeijne, G., et al., (1985), id.

A linker typically contains from 1 to 20 amino acid residues and may bepositioned between the various polypeptide sequences of the engineeredfusion protein of the invention, for example, between the S1 segment andthe CD40L segment or next to a trimerization sequence or next to aprotein tag. One such linker is the non-polar amino acid linkerdescribed by SEQ ID NO: 8 and encoded by SEQ ID NO: 7, though othernon-polar linkers may also be used, preferably alanine or othernon-polar amino acid residues.

An engineered CD40-targeted S1 fusion protein of the invention may alsoincorporate a protein tag to facilitate its isolation and purification.One such tag is a hexa-His tag (SEQ ID NO: 4), though other protein tagsused to purify proteins may also be incorporated, such as thosedescribed by SEQ ID NOS: 13-17. A protein tag may be placed at theN-terminus, the C-terminus or at another position within the body of theengineered fusion protein where it can bind to its complementarysubstrate during purification.

An engineered fusion CD40-targeted S1 fusion polypeptide of theinvention may be expressed by a chimeric polynucleotide, preferably onein a vector or DNA construct that can be transformed or transfected intoa host cell and then expressed by the host cell. Advantageously, thecoding sequences of the chimeric polynucleotide, including thoseencoding S1 and the CD40 ligand, are codon-optimized to modulate ormaximize expression in a particular host cell, such as in Vero E6 cells.Software suitable for optimizing codon usage is known and may be used tooptimize codon usage in a chimeric polynucleotide encoding an engineeredCD40-targeted MERS-CoV S1 fusion polypeptide, see Optimizer available athypertext transfer protocol://genomes._urv.cat/OPTIMIZER/ (last accessedAug. 24, 2018). Codon usage frequencies for various organisms are knownand are also incorporated by reference to the Codon Usage Database atworldwideweb.kazusa.or.jp/codon/ (last accessed Aug. 24, 2018).

An engineered fusion CD40-targeted S1 fusion polypeptide of theinvention may be incorporated into a composition, such as apharmaceutical composition or vaccine composition suitable foradministration to a subject or in a concentrated or preserved form forlater use. Such a composition typically will contain a pharmaceuticallyacceptable carrier, excipient and/or adjuvant or storage ingredients.

The engineered fusion CD40-targeted S1 fusion polypeptide of theinvention or a polynucleotide encoding it may be administered to asubject in need of prophylaxis or treatment for MERS-CoV infection. Sucha method may induce protective immunity, may reduce the risk of becominginfection, or reduce the severity of MERS-CoV infection or its symptoms,for example, this method may reduce viral titers in a subject includingthose in blood, serum, plasma, saliva, lavage fluid, sputum or otherrespiratory system secretions or in other biological samples obtainedfrom the subject. The prophylactic or therapeutic administration of apolypeptide of the invention may reduce mortality or morbidity ofMERS-CoV infection as measured by a decrease of fever, rhinorrhea (nasaldischarge), cough, malaise (lethargy), shortness of breath, vomiting,diarrhea, anorexia, sneezing or other symptoms of respiratory distress,compared to a not immunized subject or to a subject immunized with aMERS-CoV vaccine that does not target CD40. MERS-CoV presence in asample may be detected serologically or by detecting its nucleic acidsby assays known in the art, such as ELISA or RT-PCR and clinicalsymptoms evaluated by those of skill in the medical arts.

A subject to whom a prophylactic or therapeutic composition of theinvention is administered is preferably human, for example, a male orfemale child less than reproductive age, a male or female ofreproductive age, a pregnant female, or an adult. The subject may be oneat risk of exposure to MERS-CoV such as one who works in proximity toinfected humans or animals or who is exposed to animal products likemilk or meat, animal fluids such as saliva or blood, or animal wasteproducts such as urine or feces, from an animal at risk of, of that is,infected with MERS-CoV. Subjects with impaired respiratory systemsincluding pneumonia, cystic fibrosis, tuberculosis, asthma, emphysema,bronchitis, smokers, lung cancer, pneumoconiosis, and chronic bronchitisand other forms of COPD, autoimmune diseases, or those at risk of immunedysfunction or immunodeficiency may also be selected as subjects.Subjects at risk of vaccine-induced lung pathology may also be selectedas the invention does not induce the severe side-effects observed for anon-targeted S1 vaccine.

A subject to whom the fusion protein or chimeric polynucleotide of theinvention may be administered also includes non-human animals such asmembers of the Camelidae family, such as a dromedary or a Bactrian camelor llamas, alpacas, vicuñas, or guanacos. A subject may also be a pet(e.g., dog or cat), livestock (e.g., horse, cattle, goats, sheep, pigs)or wild animal (e.g., bats, mice, rats) susceptible to infection withMERS-CoV or which act as a vectors, carriers or reservoirs for thevirus. The term subject includes both adult and juvenile and male andfemale subjects. Preferably, a vaccine for use with a particular specieswill use the CD40L for that species, for example, dromedary CD40L (SEQID NO: 25) would be used for a dromedary vaccine, while human CD40L (SEQID NO: 23) would be used in an engineered CD40-targeted S1 polypeptidevaccine for humans.

Another embodiment of the invention is directed to a chimericpolynucleotide encoding an engineered CD40-targeted S1 polypeptide orother fusion polypeptide as disclosed herein. This polynucleotide may beDNA or RNA. It may form a part of a vector or DNA construct which islinear or circular. Typically, the polynucleotide will includeinitiation and termination signals operably linked to regulatoryelements, such as a promoter sequence or a polyadenylation signal andare expressible in a host cell. In some embodiments, a DNA constructwill be a vector or shuttle vector such as plasmid than can replicate ina prokaryotic cell, such as in E. coli or B. subtilis or that canreplicate in a eukaryotic cell such as a yeast, insect or mammaliancell. A shuttle vector may contain origins of replication for bothprokaryotic and eukaryotic cells permitting replication of the vector ineither kind of cell. A vector may also contain one or more expressioncontrol sequences, such as promoters including inducible promoters,enhancer sequences, or ribosome binding sites, or one or more detectablemarker sequences, such as polynucleotides that encode antibioticresistance or detectable enzymes

A vector or DNA construct of the invention may be transformed into aselected host cell which expresses the polynucleotide of the inventionto obtain a polypeptide containing the desired immunogens or epitopes,such as S1 protein determinants. One example of such a vector isnon-replicating recombinant adenovirus-5 (rAd5).

Many mammalian protein expression systems are known and commerciallyavailable including those described at to hypertext transfer protocolsecure://worldwideweb.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression.html(incorporated by reference, last accessed Aug. 24, 2018). Prokaryoticand eukaryotic host cells that may be used to express mammalianproteins, such as MERS-CoV S1 which is naturally expressed by mammaliancellular machinery, include prokaryotic cells of Escherichia coli,Corynebacgeriaum, Pseudomonas fluroescens; and eukaryotic host cellssuch as Sacharomyces cerevisiae, Pichia Pastoris, filamentous fungi,baculovirus-infected insect cells, leishmania, and a variety ofmammalian cells such as Vero E6, Chinese Hamster ovary (CHO) and Humanembryonic kidney (HEK) cells. Thus, one skilled in the art may select anappropriate vector and host cell expression system for transformationwith a polynucleotide of the invention that encodes a CD40-targeted S1fusion protein.

One embodiment of the chimeric polynucleotide of the invention is anucleic acid based vaccine and a pharmaceutically acceptable carrier,excipient or adjuvant. Immune responses against MERS-CoV may be inducedby administering a DNA- or RNA-based vaccine containing a chimericpolynucleotide encoding a CD40-targeted S1 fusion protein or otherCD40-targeted polypeptide. A DNA- or RNA-based vaccine may beadministered subcutaneously, intramuscularly, intravenously,intrapulmonarily, or intralymphatically. In one mode, the vaccine may bee complexed to particles or beads that are administered to anindividual, for example, using a vaccine gun.

Another aspect of the invention is directed to a method for reducingside effects of administering a vaccine to a subject, especially asubject at risk of or suffering from a respiratory system disease ordefect in immunity. As shown herein, non-targeted vaccination of asubject with MERS-CoV S1 antigen produced harmful effects in the lungsupon rechallenge of the vaccinated subject with MERS-CoV. The vaccinesof the invention which target S1 to antigen presenting cells bearingCD40 avoid these harmful effects. A reduction in side effects may bequantified by comparison of a CD40-targeted vaccine with an otherwiseidentical or similar non-targeted vaccine.

Another embodiment of the invention is a kit containing theCD40-targeted MERS-CoV polypeptide or nucleic acid encoding thispolypeptide as disclosed herein. The kit can be used to protect or treata subject at risk of infection or who is infected with MERS-CoV. The kitmay contain packaging materials such as sterile containers enclosing theMERS-CoV peptide, the polypeptide encoding it, an adjuvant, or apharmaceutically acceptable excipient or carrier, or mixtures thereof.It may contain devices for administering these materials to a subject,such as a syringe and needle, a device for administration to a mucousmembrane, such as an atomizer or inhaler for administration via therespiratory system, or an electroporation device for administration ofRNA or DNA via the skin. It may also contain instructions for use. Anymedium capable of storing instructions and communicating them to an enduser may be used including package inserts, such as writteninstructions, or electronic storage media (e.g., magnetic discs, tapes,cartridges), optical media (e.g., CD ROM), and the like. Theinstructions for use of the kit may also include an address of aninternet site which provides instructions.

EXAMPLES

In silico design and codon-optimization of the immunogen and generationof rAd vaccines. As described in the following example, a fusion genewas designed and codon-optimized to express a secreted and CD40-targetedconsensus S1 protein.

The S1 segment of this fusion protein was designed to express aconsensus MERS-CoV S1 subunit derived from all published MERS-CoV S1protein sequences.

All available MERS-CoV S sequences were downloaded from GenBank databaseand this data set was filtered by removing sequences containingambiguous amino acid codes. The final dataset was multiply aligned usingCLUSTALW and the Shannon entropy for each amino acid position weredetermined and the consensus sequence was then obtained for S1 subunit(amino acids 1-747, SEQ ID NO: 2). The signal peptide from MERS-CoV Sprotein was maintained at the N-terminus so that the fusion protein canbe secreted from rAd5-infected cells. The coding region for theconsensus MERS-CoV S1 subunit (coding region 1-2241) was thencodon-optimized for mammalian expression (SEQ ID NO: 2). The syntheticfusion gene was synthesized by linking the coding region of MERS-CoV S1to a polynucleotide (SEQ ID NO: 3) encoding a histidine tag sequenceHHHHHH (SEQ ID NO: 4), followed by a coding region (SEQ ID NO: 5) for a27 amino acid residue fragment from the bacteriophage T4 fibritintrimerization motif GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 6) connectedvia a coding region (SEQ ID NO: 7) for a non-polar amino acid linker AAA(SEQ ID NO: 8 where the terminal Xaas are not present) and to a codingregion (SEQ ID NO: 9) for the ectodomain of CD40L amino acid residues117-260 (SEQ ID NO: 10).

This DNA construct (SEQ ID NO: 11) expresses the fusion proteinS1/F/CD40L shown in FIG. 3 (SEQ ID NO: 12), which shows the whole codingregion for the synthetic fusion gene. The fusion gene was then used togenerate the proposed rAd construct, rAd5-S1/F/CD40L (FIG. 4A), inaddition to rAd vaccines expressing secreted and consensus S1 proteinalone (rAd5-S1)(FIG. 4B) and a vector control expressing GFP, rAd5-GFP(FIG. 4C), as shown in FIGS. 4A, 4B and 4C.

Cells and MERS-CoV virus. Vero E6 cells were cultured and maintained incomplete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%heat-inactivated fetal bovine serum (FBS), and 100 U/ml penicillin and100 μg/ml streptomycin in a humidified atmosphere of 5% CO₂ incubator.MERS-CoV-EMC/2012, was provided by Heinz Feldmann (NIH, NIAID RockyMountain Laboratories, Hamilton, Mont.) and Ron A. Fouchier (ErasmusMedical Center, Rotterdam, Netherlands). MERS-CoV amplification andculture was done in Vero E6 cells. The titer was determined by usingTCID₅₀ assay and virus stocks were stored in aliquots at −80° C. Allwork involving infectious MERS-CoV was conducted within approvedbiosafety level 3 (BSL-3) at the National Galveston Laboratory.

In vivo assessment. The immunogenicity and the protective efficacy ofthe developed rAd-based vaccines expressing the fusion proteinrAd5-S1/F/CD40L, MERS-CoV subunit 1 (S1) of the spike protein rAd5-S1 orthe control rAd vector rAd5-GFP were evaluated against MERS-CoVinfection in a human DPP4 transgenic mouse model (hDPP4 Tg⁺ mice). Asshown in FIG. 5, a total of 45 hDPP4 Tg⁺ mice aged 4-5 months were usedin this study in which 15 mice were used per group. Mice wereintramuscularly immunized twice, 28 days apart with 10⁹ of rAd candidatevaccines. Blood samples were collected 3 weeks after each immunizationto assess immunogenicity of the candidate vaccines by nAb assay. Fiveweeks post the second immunization, mice were intranasally (in)challenged with 10³ (˜100 LD₅₀) MERS-CoV (EMC2012 strain) and weremonitored for morbidly (loss in body weight) and morbidity on dailybases. On day 3 post-infection, 4 mice from each group were sacrificedto assess the virus titer and pathology in their lungs. The remainingmice were monitored for morbidly and morbidity for up to 18 days. Allanimal experiments were performed in accordance with the Guide of NIHand AAALAC and were approved by the Institutional Animal Care and UseCommittee at the University of Texas Medical Branch at Galveston, Tex.,USA. Animals were housed in on-site animal facilities at GalvestonNational Laboratory under a 12:12 light/dark cycle with room temperatureand humidity kept between 21-25° C. and 31-47%, respectively, with adlibitum access to food and water. All animal studies involvinginfectious MERS-CoV were conducted within approved animal BSL-3laboratories at the National Galveston Laboratory, strictly followingapproved notification-of-usage (NOU) and animal protocols and theguidelines and regulations of the National Institutes of Health andAAALAC.

Lung pathology. At day 3 post challenge, 4 mice in each group wereeuthanized and their lungs were collected and examined for pathologicalchanges after immunization and viral challenged. Briefly, lung tissuesobtained from necropsy samples were fixed in 10% buffered formalin for72 hrs, transferred to 70% ethanol, and later paraffin embedded.Histopathological evaluation was performed on deparaffinized sectionsstained by routine hematoxylin-eosin (H&E) staining. Evaluations forhistopathology were done by pathologists at the department of pathology,UTBM, who were masked for each specimen source. Numeric scores wereassigned to assess the extent of pathological damage.

Viral titration by TCID₅₀. Lungs collected from challenged mice on day 3post-challenge were used to determine viral titer by TCID₅₀. Briefly,pieces of collected lung tissue specimens were weighed and homogenizedin PBS containing 2% fetal calf serum (FCS) with a TissueLyser (Qiagen,Retsch, Haan, Germany). After clarification of the cellular and tissuedebris by centrifugation, the titers of the resulting suspensions ofinfected tissues were determined using TCID₅₀ assay for quantifyingyields of infectious virus. The virus titers of individual samples wereexpressed as log 10 TCID₅₀ per gram of tissue and the minimal detectablelevel of virus was 2.5 log 10 TCID₅₀ as determined by lung size.

Viral titration by RT-qPCR. Lung samples from each group of mice (n=4per group) were transferred to individual vials containing RNA latersolution and subsequently homogenized and subjected to total RNAisolation using TRIzol Reagent. To determine the viral titer in thelung, MERS-CoV-specific upstream E gene (upE) and endogenous controlgene (mouse 13-Actin) were quantified using one-step RT-PCR. Ct valuesfor each sample were analyzed against Ct values generated from astandard curve of MERS-CoV mRNA copy number. Relative MERS-CoV upE mRNAexpression value was calculated for each replicate and expressed as theequivalent of log 10 equivalents per gram (TCID eq/g) of the tissue bythe standard threshold cycle (ΔΔCT) method using Bio-Rad CFX Manager 3.0software.

Neutralizing Antibody Assays. The standard micro-neutralization assaywas used to quantify the neutralizing Abs from immunized and controlgroups. Briefly, starting at a dilution of 1:10, 60-μl volumes of serial2-fold dilutions of heat-inactivated serum specimens obtained 3 weeksafter each immunization via retro-orbital bleeding were transferred intoduplicate wells of 96-well plates containing 120 TCID₅₀ of MERS-CoV at60 μl of DMEM medium/per well, giving a final volume of 120 μl/well. Theantibody-virus mixtures were incubated at RT for 1 hr before transfer of100 μl of the mixtures (containing 100 TCID₅₀ of MERS-CoV) intoconfluent Vero E6 cell monolayers in 96-well plates. Six wells of VeroE6 cells cultured with equal volumes of DMEM medium with and withoutvirus were included in these assays as positive and negative controls,respectively. After 72 hrs of incubation, when the virus control wellsexhibited advanced virus-induced CPE, the neutralizing capacity ofindividual serum samples was assessed by determining the presence orabsence of CPE. Reciprocals of the last dilutions of serum specimenscapable of completely preventing the formation of CPE in 50% of thewells were used as the neutralizing antibody titers and expressed as 50%neutralizing titers (MNT₅₀).

Data analysis. Statistical analysis was conducted using one-way ANOVA.Bonferroni's post-test was used to adjust for multiple comparisonsbetween the different groups. All statistical analysis was conductedusing GraphPad Prism software (San Diego, Calif.). P values <0.05 wereconsidered significant.

A single dose of CD40-targeted MERS-CoV S1 elicited high levels of nAbsin mice. In order to evaluate the immunogenicity of our vaccinecandidates, we immunized mice i.m. with two doses of the generatedvaccines and measured the levels of nAbs before and 3 weeks after eachimmunization. All mice from all groups showed no detectable levels ofnAbs in pre-bleed samples collected before immunization as expected. Asshown in FIG. 6A, mice immunized with a single does of eitherrAd5-S1/F/CD40L or rAd-S1 elicited high levels of nAbs compared tocontrol group (rAd-GFP), but only rAd5-S1/F/CD40L induced highlysignificant levels of nAbs against live MERS-CoV virus, suggesting thata single dose of rAd5-S1/F/CD40L could result in a strong humoralresponse and neutralizing activity when compared with rAd-S1. Afterboosting, all animals from both groups rAd5-S1/F/CD40L and rAd-S1developed robust and significant levels of nAbs (FIG. 6B), indicatingthat at least two doses of rAd-S1 were required to induce levels of nAbssimilar to those obtained by a single dose of rAd5-S1/F/CD40L.Furthermore, evaluation of circulating levels of S1-specific total IgGafter one or two doses by ELISA also showed significantly higher levelsin animals immunized with rAd5-S1/F/CD40L compared to rAd-S1 group (datanot shown). These findings confirm that using CD40L as molecularadjuvant enhances the immunogenicity of S1 based vaccines and mayrepresent a very promising vaccine platform to induce protectiveimmunity with a single dose.

CD40-targeted MERS-CoV S1 protected mice from MERS-Co V withoutvaccine-associated lung pathology. To this end, immunized mice werechallenged with 100 LD₅₀ of MERS-CoV in order to investigate theprotective efficacy of the vaccines. Monitoring challenged animals for 3weeks showed that both rAd5-S1 or rAd5-S1/F/CD40L immunized groups werecompletely protected (FIG. 7A) and maintained their weight (FIG. 7B),suggesting that both vaccines are protective in this highly MERS-CoVpermissive mouse model and could represent potential vaccine candidates.

Evaluation of lung pathology in immunized and challenged mice on day 3post-challenge showed that there was up to 15% of multiple monocytic andlymphocytic infiltrations in the lung of rAd5-GFP group compared toother groups which showed minimal or no lung infiltration (Table 1 andFIGS. 8A-8F).

TABLE 1 Viral titer and lung pathology. Viral titer Lung pathologyVaccine Animal (log₁₀ TCID₅₀/g)^(a) (Grades 0-3)^(d) rAd5-empty 1 4.16 12 3.67 1 3 3.51 1 4 4.58 0 3.98 ± 0.24^(b) rAd5-S1 1  ND^(c) Hemorrhage2 ND Hemorrhage 3 ND Hemorrhage 4 ND Hemorrhage rAd5-S1/F/CD40L 1 ND 0 2ND 0 3 ND 0 4 ND 0-1^(e) ^(a)rAd5-S1 and rAd5-S1/F/CD40L reducedMERS-CoV replication in the lungs of infected mice significantly (p =0.015). ^(b)=mean ± Sd. ^(c)ND: not detected (below the detection limitof 2.5 log₁₀). ^(d)Grades 0-3: 0-normal/no pathology; 1-up to 15%pathology; 2-up to 25% pathology; 3-35% of the entire lung section withmultiple monocytic and lymphocytic infiltrates. ^(e)only small area withfew infiltrates.

Surprisingly, while rAd5-S1 immunization protected animals from deathand weight loss, and prevented lung infiltration similar torAd5-S1/F/CD40L, the inventor observed perivascular hemorrhage in thewhole lung of all examined mice (FIG. 8B, 8E). This is consistent withnon-CD40-targeted S1 immunogen leading to vaccine-induced lungpathology, here, as characterized by pervascular hemorrhage.

On the other hand, immunization with rAd5-S1/F/CD40L protected mice tosimilar levels as rAd5-S1 but without such hemorrhage, indicating thatusing CD40L as targeting molecule and molecular adjuvant does not onlyenhance immunogenicity but also prevents vaccine-associated pulmonarypathology. Such vaccine-associated pathologies include interstitial lungdisease, priming for a more serious enhanced MERS-CoV respiratorydisease, hypersensitivity to MERS-CoV antigens, as well as otherundesired inflammatory or immunological responses triggered oraggravated by vaccination and subsequent exposure to virus or viralantigens.

Both MERS-CoV S1-based vaccines prevented pulmonary viral replication.To further confirm that the immunized mice were protected from MERS-CoVinfection after challenge, we measured lung viral titer on day 3post-challenge by TCID₅₀ and RT-qPCR. Analysis of lung viral titer byTCID₅₀ confirmed that both rAd5-S1/F/CD40L or rAd5-S1 vaccines preventedviral replication at a significant levels in the lungs of immunized micecompared to control animals to levels below the detection limits ofTCID₅₀ assay (Table 1). Similarly, measurement of viral RNA in lungsdemonstrated that both groups immunized with either rAd5-S1/F/CD40L orrAd5-S1 had significantly lower viral loads compared to the rAd-GFPcontrol group by 3-4 logs as shown in FIG. 9. Although there was nosignificant difference between the viral load levels betweenrAd5-S1/F/CD40L and rAd5-S1, the viral load was lower by 1 log inrAd5-S1/F/CD40L immunized mice compared to rAd5-S1 group consistent withthe overall stronger immune response we observed in this animal group.

The rapid spread and persistence of MERS-CoV in the Arabian Peninsula inaddition to the associated high mortality rates represent a seriousglobal public health concern especially that the elimination of thezoonotic reservoir is impossible. This threat is further complicated bythe absence of prophylactic or therapeutic measures. Therefore,development of safe and preventive vaccine is urgently needed. Severalgroups have investigated various MERS-CoV vaccine platforms to combatMERS-CoV. See Wang et al.; Muthumani et al.; Coleman et al.; Du et al.;Al-Amri et al.; Ma et al.; Song et al.; Haagmans et al.; Guo et al.; Kimet al.; Alharbi et al.; and Agrawal et al., each incorporated herein byreference in their entirety. Most of these experimental vaccines werebased on MERS-CoV full-length or truncated versions of the spikeprotein; these prototype vaccines induced high levels of nab andsometimes conferred complete protection against MERS-CoV challenge inseveral animal models. However, serious safety concerns are associatedwith vaccines for several CoVs including MERS-CoV and need to beelucidated and better understood. See Agrawal et al.; Weingartl et al.;Tseng et al.; Yang et al.; Czub et al.; Deming et al.; Olsen et al.;Jaume et al.; and Weiss et al; each incorporated herein by reference intheir entirety.

As shown by the Example, both rAd expressing S1 or CD40-targeted S1induced significant levels of anti-MERS-CoV systemic IgG and nAbs.However, the use of uCD40L as molecular adjuvant and targeting moleculeenhanced the immunogenicity of S1 to the extent that one dose wassufficient to elicit significant levels of nAbs compared to controlgroups. Advantageously, both vaccines provided complete protection andprevented and/or minimized pulmonary viral replication and monocytic andlymphocytic lung infiltration in immunized mice compared to controlgroup immunized with rAd5-GFP vector.

Surprisingly, it was found that mice immunized with the CD40L-targetedvaccine did not experience the severe perivascular hemorrhaging of themice receiving the untargeted MERS-CoV S1 vaccine. As shown by FIG. 8,all examined mice receiving untargeted S1 (without CD40L) showed severeperivascular hemorrhage in the whole lung after viral challenge despitethe observed robust immune response and protection. These results showthat a CD40-targeted vaccine is safer than an otherwise similar S1vaccine that is not targeted to CD40 on antigen presenting cells.

The inventor has shown that immunogens such as MERS-CoV S1 antigen maybe targeted to CD40-expressing antigen presenting cells using aCD40L-type ligand and that such targeting enhances both immunogenicityand protective efficacy against virus challenge, but also minimizes therisk of side-effects such as respiratory pathology associated with theadministration of non-targeted MERS-CoV vaccines. These results show thegeneral applicability of this approach to production of vaccines toMERS-CoV as well as other kinds of viruses.

Terminology. Terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

The headings (such as “Background” and “Summary”) and sub-headings usedherein are intended only for general organization of topics within thepresent invention, and are not intended to limit the disclosure of thepresent invention or any aspect thereof. In particular, subject matterdisclosed in the “Background” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

Links are disabled by deletion of http: or by insertion of a space orunderlined space before www. In some instances, the text available viathe link on the “last accessed” date may be incorporated by reference.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all sub-ranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the materials, compositions, devices,and methods of this technology. Similarly, the terms “can” and “may” andtheir variants are intended to be non-limiting, such that recitationthat an embodiment can or may comprise certain elements or features doesnot exclude other embodiments of the present invention that do notcontain those elements or features.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

The invention claimed is:
 1. A Middle East respiratory syndromecoronavirus (MERS-CoV) vaccine, comprising: at least one carrierselected from the group consisting of a polysorbate composition, acarbohydrate, and a protein, an aqueous buffer solution, and apolypeptide that comprises at least one receptor-binding domain,immunogenic fragment, or epitope of S1 polypeptide of MERS-CoV, at leastone trimerization motif, and at least one ligand for CD40; wherein theat least one trimerization motif comprises the amino acid sequence ofSEQ ID NO: 6; or wherein the CD40 ligand is human CD40L comprising thesequence of SEQ ID NO: 23 or a sequence that is at least 70% identicalthereto, or a CD40-binding fragment thereof; or wherein the CD40 ligandis camelid or human CD40L comprising the sequence of SEQ ID NO: 25 or 27or a sequence that is at least 70% identical thereto, or a CD40-bindingfragment thereof.
 2. The MERS-CoV vaccine of claim 1, wherein the atleast one receptor-binding domain, immunogenic fragment, or epitope ofS1 polypeptide of MERS-CoV comprises an amino acid sequence that is atleast 70% identical to the amino acid sequence of SEQ ID NO:
 2. 3. TheMERS-CoV vaccine of claim 1, wherein the at least one receptor-bindingdomain, immunogenic fragment, or epitope of S1 polypeptide of MERS-CoVcomprises a sequence of SEQ ID NO:
 2. 4. The MERS-CoV vaccine of claim1, wherein the at least one trimerization motif comprises the amino acidsequence of SEQ ID NO:
 6. 5. The MERS-CoV vaccine of claim 1, whereinthe CD40 ligand is human CD40L comprising the sequence of SEQ ID NO: 23or a sequence that is at least 70% identical thereto, or a CD40-bindingfragment thereof.
 6. The MERS-CoV vaccine of claim 1, wherein the CD40ligand is camelid or human CD40L comprising the sequence of SEQ ID NO:25 or 27 or a sequence that is at least 70% identical thereto, or aCD40-binding fragment thereof.
 7. The MERS-CoV vaccine of claim 1 thatfurther comprises at least one peptide linker ranging from 1 to 10 aminoacid residues in length between the at least one immunogenic fragment,receptor-binding domain, or epitope of S1 polypeptide of MERS-CoV,and/or the at least one trimerization motif, and/or the at least oneligand for CD40.
 8. The MERS-CoV vaccine of claim 1, comprising acarrier selected from the group consisting of sorbitol, mannitol,starch, sucrose, dextran, glutamate, and glucose.
 9. The MERS-CoVvaccine of claim 1, comprising an albumin carrier.